Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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Development of Molecular Biological Methods to Analyze Bacterial Species Diversity in Freshwater and Soil Ecosystems

  • Lee, Dong-Hun (Department of Microbiology and Research Institute for Genetic Engineering, Chungbuk National University) ;
  • Noh, Sung-Ae (Department of Microbiology and Research Institute for Genetic Engineering, Chungbuk National University) ;
  • Kim, Chi-Kyung (Department of Microbiology and Research Institute for Genetic Engineering, Chungbuk National University)
  • Published : 2000.03.01
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Abstract

A new method was developed for the rapid analysis of diverse bacterial species in the natural environment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indigenous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats.

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References

  1. Microbiol. Rev. v.59 Phylogenetic identification and in situ detection of individual microbial cells without cultivation Amann, R.I.;W. Ludwig;K.H. Schleifer
  2. Adv. Microb. Ecol. v.7 Diversity of microbial communities Atlas, R.M.
  3. Limnal. Oceanogr v.38 Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblaes DeLong, E.F.;D.G. Franks;A.L. Alldredge
  4. Nature 356 Novel major archaebacterial group from marine plankton Fuhrman, J.A.;K. McCallum;A.A. Davis
  5. Microbiol. Rev. v.58 Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective Gutell, R.R.;N. Larsen;C.R. Woese
  6. PCR Methods Appl. v.1 PCR-SSCP: A simple and sensitive method for detection of mutations in te genomic DNA Hayashi, K.
  7. Appl. Environ. Microbiol. v.62 Non-radioactive method to study genetic profiles of natural bacerial communities by PCR-single-strand-conformation polymorphism Lee, D.-H;Y.-G Zo;S.-J. Kim
  8. Mol. Cells 5 A non-radioactive method for simultaneous detection of single-strand conformation polymorphisms (SSCPs) and heteroduplexes Lee, S.-T;S.-K. Park;K.-H. Lee;S.A. Holmes;R.A. Spritz
  9. J. Baceriol. v.174 Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment Liesack, W.;E. Stackebrandt
  10. Modern soil microbiology Microbial diversity in soil: the need for a combined approach using molecular and cultivatin techniques Liesack, W.;P.H. Janssen;F.A. Rainey;N.L. Ward-Rainey;E. Stackebrandt;J.D. van Elsas(ed.);J.T. Trevors(ed.);E.M.H. Wellington(ed.)
  11. Appl. Environ. Microbiol. v.63 Characterizatin of microbial diversit by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA Liu, W.-T.;T.L. Marsh;H. Cheng;L.J. Forny
  12. Nucleic Acids. Res. v.27 A new version of the RDP (Ribosomal Daabase Project) Maidak, B.L.;J.R. Cole;C.T. Parker;G.M. Garrity;N. Larsen;B. Li;T.G. Lilburn;M.J. McCaughey;G.J. Olsen;R. Overbeek;S. Pramanik;T.M. Schmidt;J.M. Tiedje;C.R. Woese
  13. Appl. Environ. Microbiol. v.59 Survival of nonculturable Aeromonas salmonicida in lake water Morgan, J.A.;G. Rhodes;R.W. Pickup
  14. Limnol. Oceanogr v.40 Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterriplankton communities Mullins, T.D.;T.B. Britschgi, R.L. Krest;S.J. Giovanoni
  15. Appl. Environ. Microbiol. v.59 Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA Muyzer, G.;E.C. De Waal;A.G. Uitterlinden
  16. Nucleic Acids Res. v.21 Compilation of small ribosomal subunit RNA structures Neefs, J.M.;Y Van de Peer;P. De Rijk;S. Chapelle;R. De Wachter
  17. Appl. Environ. Microbiol. v.61 Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment Oliver, J.D.;F. Hite;D. McDougald;N.L. Andon;L.M. Simpson
  18. FEMS Microbiol. Letters 100 DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities Rochelle, P.A.;J.C. Fry;R.J. Parkes;A.J. Weightman
  19. J. Bacteriol. v.173 Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing Schmidt, T.M.;E.F. DeLong;N.R. Pace
  20. Appl. Environ. Microbiol. v.64 A new approach to utilize PCR-Single-Strand-Conformation polymorphism for 16S rRNA gene-based microbial community analysis Schwieger, F.;C.C. Tebbe
  21. Bio echniques 22 Separation of DNA strans facilitates detection of point mutations by PCR-SSCP Selvakumar, N.;B.-C. Ding;S.M. Wilson
  22. Genomics 16 The sensitvity of single-strand conformation polymorphism analysis for the detection of single base substitutions Sheffield, V.C.;J.S. Beck;A.E. KWitek, D.W. Sandstrom;E.M. Stone
  23. Int. J. Syst. Bacteriol. Approved Lists of Bacterial Names Skerman, V.B.D.;V. McGowan;P.H.A. Sneath
  24. Appl. Environ. Microbiol. v.56 High diversity in DNA of soil bacteria Torsvik, V.;. Goksoyr;F.L. Daae
  25. Adv. Microb. Ecol. v.12 Ribosomal RNA anlaysis of microorganisms as they occur in nature Ward, D.M.;M.M. Beteson;R. Weller;A.L. Ruff-Roberts
  26. Appl. Environ. Microbiol. v.64 Molecular detectin, isolation, and physiological characterization of functionally dominant phenoldegrading baceria in activated sludge Watanabe, K;M. Teramoto;H. Futamata;S. Harayama
  27. PCR technology: current innovations Non-isotopic single-strand conformation polymorphism (SSCP) analysis of PCR products Yap, E.P.H.;J.O. McGee;H. G. Griffin(ed.);A. M. Griffin(ed.)